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Circulation Journal
Official Journal of the Japanese Circulation Society
http://www. j-circ.or.jp

Arterial 18F-Fluorodeoxyglucose Uptake Reflects Balloon
Catheter-Induced Thrombus Formation and Tissue
Factor Expression via Nuclear Factor-κB
in Rabbit Atherosclerotic Lesions
Atsushi Yamashita, MD, PhD; Yan Zhao, MD, PhD; Songji Zhao, MD, PhD; Yunosuke Matsuura, MD;
Chihiro Sugita, BSc; Takashi Iwakiri, MD; Nozomi Okuyama, BSc; Kazuyo Ohe, PhD;
Chihiro Koshimoto, PhD; Keiichi Kawai, PhD; Nagara Tamaki, MD, PhD;
Yuji Kuge, PhD; Yujiro Asada, MD, PhD

Background: Imaging modalities to assess atherosclerotic plaque thrombogenicity have not been established, so
in this study the relationship between [18F]-fluorodeoxyglucose (18F-FDG) uptake and thrombus formation was investigated in rabbit atherosclerotic arteries.
Methods and Results: Atherosclerotic plaque was induced in the iliacofemoral artery by balloon injury and a 0.5%cholesterol diet. At 3 weeks after the first balloon injury, the arteries were visualized by 18F-FDG positron emission
tomography (PET) imaging 2 h after an 18F-FDG infusion, and then arterial thrombus was induced by a second balloon injury of both iliacofemoral arteries. Imaging with 18F-FDG-PET revealed significantly more radioactivity along
the injured (0.63±0.12 SUVmax), than the contralateral non-injured artery (0.34±0.08 SUVmax, n=17, P<0.0001).
Arterial radioactivity measured by autoradiography positively correlated with macrophage area, the number of nucleithat were immunopositive for nuclear factor κ B (NF-κB), and tissue factor (TF) expression. The immunopositive
areas for glycoprotein IIb/IIIa and fibrin in thrombi were significantly larger in the atherosclerotic than in the contralateral arteries, and significantly correlated with radioactivity in PET (r=0.92, P<0.001, n=10) and autoradiography(r=0.73, P<0.0001, n=50) in the arteries. Inhibition of NF-κB significantly reduced TF expression in cultured atherosclerotic plaque.
Conclusions: Arterial 18F-FDG uptake reflects the thrombogenicity of atherosclerotic plaque following balloon injury.
Key Words: Atherothrombosis; 18F-FDG; Nuclear factor-κB; Rabbits; Tissue factorThrombus formation is a major complication of atherosclerosis, but although disruption of atherosclerotic
plaques is recognized as a trigger of atherothrombosis,
not all thrombi result in complete luminal occlusion and symptomatic events.1,2 Therefore, thrombus size is critical to theonset of clinical events. Thrombus formation and propagation
are regulated by many factors, such as vascular wall thrombogenicity, local hemorheology, blood thrombogenicity, and fibrinolytic activity.3 Among these, the thrombogenicity of ath-

erosclerotic plaque is an essential factor in atherothrombosis.
Tissue factor (TF) is an initiator of the coagulation cascade thatis expressed in the adventitia and to various degrees in the
media of normal arteries.4 Because atherosclerotic plaques
express TF, the degree of TF expression is a crucial determinant of plaque thrombogenicity.5 Although current imaging
modalities support high-quality plaque characterization,6 thosethat can assess atherosclerotic plaque thrombogenicity have notbeen established.

Received November 27, 2012; revised manuscript received April 22, 2013; accepted May 23, 2013; released online July 6, 2013   Time forprimary review: 29 days
Department of Pathology (A.Y., Y.M., C.S., T.I., N.O., Y.A), Department of Internal Medicine (Y.M., T.I.), Department of Faculty of
Medicine, Frontier Science Research Center (C.K.), University of Miyazaki, Miyazaki; Department of Tracer Kinetics & Bioanalysis(Y.Z., S.Z.), Department of Nuclear Medicine (N.T.), Graduate School of Medicine, Central Institute of Isotope Science (Y.K.), Hokkaido
University, Sapporo; Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University,
Ishikawa (K.O., K.K.); and Biomedical Imaging Research Center, University of Fukui, Fukui (K.K.), Japan
Mailing address: Yujiro Asada, MD, PhD, Department of Pathology, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake,Miyazaki 889-1692, Japan.   E-mail: yasada@med.miyazaki-u.ac.jp
ISSN-1346-9843  doi: 10.1253/circj.CJ-12-1463
All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: cj@j-circ.or.jp

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YAMASHITA A et al.

Figure 1.   Experimental protocol for the animal studies of [18F]-fluorodeoxyglucose (18F-FDG) uptake and thrombus formation in
rabbit atherosclerotic arteries (n=5 Japanese white rabbits). ARG, autoradiography; IHC, immunohistochemistry.

Positron emission tomography (PET) imaging using [18F]fluorodeoxyglucose (18F-FDG) has been advocated as a meansof evaluating arterial inflammation.7 The uptake of 18F-FDG
closely correlates with plaque macrophage contents in animalmodels,8–10 which suggests that the degree of 18F-FDG accumulation in the vessel wall reflects underlying levels of inflammation. Clinical studies have also identified a relationship between 18F-FDG uptake and numbers of cardiovascular risk
factors, as well as risk for future events.11–13 The uptake of
18F-FDG is significantly higher in aortic segments with, than
without thrombus in a rabbit model of advanced atherosclerosis.14 These lines of evidence imply an association between
18F-FDG uptake and arterial thrombus formation, although theunderlying mechanism is unknown. We investigated this notion in a rabbit model of atherosclerosis.

Methods
Experimental Protocol
The Animal Care Committee of Miyazaki University and
Hokkaido University approved the animal research protocolsof the present study, which also conformed to the Guide for
the Care and Use of Laboratory Animals published by the US
National Institutes of Health.
Figure 1 shows the experimental protocol of the 18F-FDGPET study. The 5 male Japanese white rabbits weighing 2.5–
3.0 kg were fed with a 0.5% cholesterol diet, and the iliacofemoral artery was injured with a balloon catheter to induce
atherosclerotic lesions. At 3 weeks after the first balloon injury, 18F-FDG-PET imaging proceeded 2 h after 18F-FDG infusion. Arterial thrombus was induced by a second balloon injury to both iliacofemoral arteries and 15 min later, the amountof radioactivity in the arteries was evaluated and the arteries
were immunohistochemically analyzed.
Atherosclerosis and Thrombosis Model
Surgery proceeded under aseptic conditions and general anesthesia was achieved via an intravenous injection of pentobarbital (25 mg/kg). At 1 week after feeding with a 0.5% cholesterol diet, an angioplasty balloon catheter (diameter, 2.5 mm;
length, 9 mm; Quantum, Boston Scientific, Galway, Ireland)
was inserted using a 4F introducer sheath (Goodman, Nagoya,Japan) via the carotid artery into the right iliacofemoral arteryunder fluoroscopic guidance to induce an atherosclerotic lesionin the artery. The catheter was inflated to 1.5 atm (balloon-toartery ratio; 1.1:1 to 1.2:1) and retracted 3 times to denude theendothelium.
After 18F-FDG-PET imaging, a 2F balloon catheter (Baxter
Healthcare) was inserted via the anterior tibial arteries into both
iliacofemoral arteries, inflated to 1.4 atm, and retracted twice toinduce arterial thrombus;15 15 min later, the rabbits were injected with heparin (500 U/kg, i.v.) and then killed 5 min laterwith an overdose of pentobarbital (60 mg/kg, i.v.). The animals were perfused with 50 ml of cold 0.01 mol/L of phosphate-buffered saline to remove residual blood from the arteries for subsequent autoradiography and immunohistochemicalevaluation.
PET Imaging and Analysis

18F-FDG-PET imaging proceeded as described with minor

modifications.16 The rabbits (n=5) were fasted for 4 h prior to
PET scanning, infused with 18F-FDG (average, 193 MBq/rabbit)
2 h prior to imaging, and anesthetized by 1.5–2.0% isoflurane
inhalation. The injected dose of 18F-FDG was determined inconsideration of the half-life of 18F (110 min). After urinary
bladder lavage with warmed saline, the rabbits were placedsupine on a customized acrylic bed in a small animal PET scanner (Inveon, Siemens Medical Solutions USA, Knoxville, TN,
USA) and scanned (list-mode acquisition) for 40 min. Body
temperature (37.4±0.2°C) was maintained using cotton bedding prepared during the PET scanning. The data were reconstructed and corrected for attenuation and scatter using 2D filtered back-projection (FBP) and maximum a posteriori (MAP)
algorithm. The spatial resolution was 1.63 mm full-width at
half-maximum (FWHM) in FBP and 1.05 mm FWHM in MAP.
The former was used to quantify the radioactivity and the latterfor visual observations. The image matrix was 256×256×159,
resulting in a voxel size of 0.385×0.385×0.796 mm. Coronal,
axial and sagittal images were visually graded. Coronal images
were quantified by centering regions of interest (ROIs) over theiliacofemoral arteries, then 3 or 4 circular ROIs (10 pixels indiameter) in the FBP images were placed on each injured andcontralateral non-injured artery. The results are expressed as
standardized uptake value (SUV). The maximum values of
SUV (SUVmax) for the bilateral arteries were confirmed andrepresented the 18F-FDG uptake in the PET images.
Radioactivity of Tissue and Arterial Slices
The amount of radioactivity in the iliacofemoral arteries, skeletal muscle tissue, and blood was measured using a well-typeγ-counter (1480 Wizard 3; Wallac Co Ltd, Turku, Finland).
The results are calculated as (%ID/g)×kg and then converted

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Arterial 18F-FDG Uptake and Thrombogenicity

to SUV.17
The arteries were cut into 5 sections that were embedded in
Tissue-Tek (Sakura, Tokyo, Japan) and frozen. Consecutive
10- or 5-μm slices were prepared for autoradiographic or histologic analysis, respectively. The 10-μm cryostat sections
were exposed to phosphor imaging plates (Fuji Imaging Plate
BAS-SR 2025, Fuji Photo Film Co Ltd, Tokyo, Japan) for 12 h,
together with a set of calibrated standards.8 The imaging plates
were then scanned using a Fuji Bio-imaging Analyzer BAS5000 with an internal resolution of 25 μm (Fuji Photo Film CoLtd) and the images were examined using image analysis
software (Multi Gauge Ver. 3.0, Fuji Photo Film Co Ltd). The
amount of radioactivity in each image is expressed as photostimulated luminescence per unit area (PSL=a×D×t, where a is
a constant, D is the amount of radioactivity exposed on theimage plate, and t is exposure time). Each PSL value/mm2 ofarterial tissue was recorded and converted to a ratio (%) of theactivity of the standard injected dose/mm2 of lesion area (%ID/mm2). The data were normalized with the animal’s bodyweight (%ID×kg/mm2) and then converted to SUV.18
Histologic Analysis
Consecutive 5-μm slices were fixed in acetone, stained withhematoxylin–eosin, and immunohistochemically examinedusing antibodies against muscle actin (HHF35, Dako, Glostrup,
Denmark), rabbit macrophages (RAM11, Dako), CD163 (AM3K, Trans Genic Inc, Kobe, Japan), nuclear factor κ B (NF-κB;
Abnova, Taipei, Taiwan), TF (mouse IgG2b subclass for rabbit
TF extracellular domain (193Ser-207Cys)), glycoprotein (GP)IIb-IIIa (Affinity Biologicals Inc, Hamilton, CA, USA) andrabbit fibrin (Takeda Chemical Industries Ltd, Osaka, Japan).
The sections were stained with Envision (Dako) or donkey
anti-sheep IgG secondary antibody (Jackson ImmunoResearch,
Baltimore, MA, USA). Horseradish peroxidase activity wasvisualized using 3, 3’-diaminobenzidine tetrahydrochloride,and counterstained with Meyer’s hematoxylin. Immunostaining controls included non-immune mouse IgG or non-immune
sheep serum instead of the primary antibodies. Areas of positive immunostaining in the vessel and thrombus were analyzed
using a color imaging morphometry system (Win Roof, Mitani,
Fukui, Japan).15 The data are expressed as ratios of the immunopositive area (μm2), or as numbers of immunopositive nuclei/mm2.
For double immunofluorescence, antibodies for rabbit macrophage (RbM2, TransGenic Inc) and smooth muscle actin(SMA; 1A4, Novus Biologicals, Littleton, CA, USA) were labeled with Mix-n-Stain CF594 antibody labeling kit (Biotium,
Hayward, CA, USA). The sections were stained with CF488A
labeled secondary antibody (Biotium) for NF-κB and CF594labeled antibodies for rabbit macrophages or SMA.
Tissue Culture of Rabbit Atherosclerotic PlaqueAtherosclerotic lesions were induced as described. The rabbits
were injected with heparin (500 U/kg, i.v.), killed 5 min laterwith an overdose of pentobarbital (60 mg/kg, i.v.) and perfusedwith 50 ml of 0.01 mol/L of phosphate-buffered saline andDulbecco’s modified Eagle’s medium. The iliacofemoral arterywas excised and the atherosclerotic plaque was separated fromthe media and adventitia. The plaque was divided into 3-mm
segments and placed in incubation medium containing 10%
fetal bovine serum in multidishes for 6 h. Cell viability wasevaluated in plaque segments stained with nicotinamide adenine dinucleotide-hydrogen (NADH) and nitro blue tetrazolium. The plaque segments (n=5) were frozen before or 6 h after
incubation, and then 5-μm sections were incubated at 37°C for

1 h in 10 ml of Gomeri Tris-HCl buffer (pH 7.4) containing
24 mg of nitroblue tetrazolium (Nacalai Tesque, Kyoto, Japan)
and 10 mg of NADH (CalBiochem, San Diego, CA, USA).19
The amount of TF protein in plaque segments was measured
using a rabbit TF ELISA kit (Uscn Life Science Inc, Wuhan,
China) before and at 6 h after incubation with Bay 11–7085(an inhibitor of inhibitor of κB kinase, Merck, Darmstadt,
Germany) or dimethyl sulfoxide.
Statistical Analysis
All data are presented as means and standard deviation. Differences between individual groups were tested using theMann-Whitney U-test (GraphPad Prizm 4.03, GraphPad Software Inc, San Diego, CA, USA). Relationships between factors were evaluated using Spearman’s rank correlation coefficient and P<0.05 was considered statistically significant.

Results
PET Imaging and Radioactivity of Arteries
Figure 2 shows the 18F-FDG-PET coronal MAP image andradioactivity accumulation before arterial thrombus formation.
The accumulation of 18F-FDG is greater along the injured than
the non-injured artery (Figure 2A). We compared 18F-FDG
uptake between the injured and non-injured arteries by analyzing coronal FBP images after drawing ROIs centered over theiliacofemoral arteries. The maximal amount of radioactivity in
the ROIs was significantly higher in the injured than in the
non-injured arteries (0.63±0.12 vs. 0.34±0.08 SUVmax; n=17,
P<0.0001; Figure 2B). Significantly more radioactivity was
found in the excised injured but not the non-injured iliacofemoral artery (1.32±0.55 vs. 0.35±0.11 SUVmean; n=5, P<0.01;
Figure 2C). Figure 2D shows that level of 18F-FDG uptake
correlated between PET image and tissue (r=0.71, P<0.05;n=10). Levels of 18F-FDG uptake were similar among the noninjured artery, blood and skeletal muscle tissue (0.35±0.11,
0.28±0.05 and 0.37±0.24 SUV, respectively; mean, n=5).
Autoradiographic and Histologic Findings
Figures 3A and 3B shows the autoradiographic and histologic
images of the injured and non-injured arteries with thrombi.
Significantly more radioactivity was found in sections of the
injured than the non-injured artery (7.96±3.84 vs. 1.05±0.93
SUV mean, n=25; P<0.0001). We evaluated cellular components, inflammatory response, vascular wall thrombogenicity
and thrombus content by immunohistochemical staining formuscle actin, RAM11 (macrophage), CD163 (M2 macrophage
marker), NF-κB, TF, GPIIb/IIIa and fibrin. The cellular composition at 3 weeks before injured arteries was rich in macrophages with smooth muscle cells (SMCs) (ie, atherosclerotic),
whereas that in the contralateral artery mainly comprised SMCs
with a few macrophages in the adventitia (Figures 3A,B).
The CD163-immunopositive area accounted for 0.2% in theatherosclerotic arteries. The ratio of CD163 positive area to
RAM11 positive area was 0.01. The ratio of immunopositive
areas for muscle actin and macrophages to vascular area wassignificantly smaller and larger, respectively, in the atherosclerotic than in the contralateral arteries (Figure 3B).
Thrombi that developed within 15 min of balloon injury
comprised platelets and fibrin (Figure 3A). More areas were
immunopositive for GPIIb/IIIa and/or fibrin in the atherosclerotic (GPIIb/IIIa, 0.030±0.019 mm2; fibrin, 0.021±0.019 mm2;
GPIIIb/IIIa+fibrin, 0.052±0.035 mm2) than in the contralateral (GPIIb/IIIa, 0.010±0.002 mm2; P<0.0001, n=25; fibrin,0.005±0.003 mm2; P<0.0001, n=25; GPIIIb/IIIa+fibrin, 0.010±

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YAMASHITA A et al.

Figure 2.  Coronal 18F-FDG-PET
image and radioactive accumulationin rabbit iliacofemoral artery. PET imaging began 120 min after 18F-FDG
injection and lasted 40 min. (A) Coronal 18F-FDG-PET image shows greater accumulation in the side with(arrows) than without (arrowheads)
balloon injury. UB, urinary bladder.
(B) Maximum radioactivity of ROIs
centered over arteries (n=17 each).
(C) Radioactive accumulation in excised arteries (n=5 each). (D) Radioactive correlation between PET images and excised arteries. 18F-FDG,[18F]-fluorodeoxyglucose; PET, positron emission tomography; ROI, region of interest; SUV, standardized
uptake value.

0.003 mm2, P<0.0001; n=25) arteries (Figure 3B).
Immunoreactivity for NF-κB and TF was mainly localized in the macrophage-rich area in the atherosclerotic artery(Figure 4A). Double immunofluorescence showed NF-κBimmunopositive nuclei in macrophages and SMCs (Figure 4B).
The ratio of macrophage to NF-κB-positive nuclei (69±13%,n=13) was more than the ratio of SMCs to them (26±9%, n=13,
P<0.0001). Immunopositive nuclei for NF-κB and the immunopositive area for TF were greater in the atherosclerotic (NFκB: 208±225/mm2, TF: 4.7±3.3%) than in the contralateral (NFκB, 22±33/mm2, P<0.0001, n=25; TF, 0.4±0.4%, P<0.0001,n=25) artery (Figure 4C). In addition, the number of NF-κB
positive nuclei and TF immunopositive areas significantly correlated (r=0.63, P<0.0001, n=50; Figure 4C).
Correlation Between 18F-FDG Uptake and Histologic
Findings
Figure 5 shows the correlation between 18F-FDG uptake andvascular/thrombus factors. Autoradiographic 18F-FDG uptake
positively correlated with areas that were immunopositive(%) for macrophages (r=0.71, P<0.0001, n=50), TF (r=0.64,
P<0.0001, n=50), and the number of NF-κB (r=0.85, P<0.0001,n=50), and negatively with SMCs (r=–0.44, P<0.01, n=50)(Figure 5A). The 18F-FDG uptake also positively correlatedwith areas that were immunopositive for GPIIb/IIIa (r=0.77,
P<0.0001, n=50), fibrin (r=0.66, P<0.0001, n=50), GPIIb/IIIa
and fibrin (r=0.73, P<0.0001, n=50; Figure 5B). In addition,
maximal 18F-FDG uptake in PET images positively correlatedwith areas that were immunopositive for GPIIb/IIIa (r=0.82,
P<0.01, n=10), fibrin (r=0.95, P<0.001, n=10), GPIIb/IIIa andfibrin (r=0.92, P<0.001, n=10; Figure 5C).
TF Expression in Tissue Cultures of Atherosclerotic PlaqueWe assayed cultured plaques to determine the associations
between NF-κB and TF expression in rabbit atherosclerotic
lesions. Atherosclerotic lesions were cut into 2-mm sections
and cultured in conditioning medium with 10% FBS. The number of NF-κB-immunopositive nuclei and the TF protein levels in the plaques were significantly increased 6 h later, compared with before plaque culture. Inhibiting NF-κB using Bay

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Arterial 18F-FDG Uptake and Thrombogenicity

Figure 3.   Autoradiographic (ARG) and histologic findings of atherosclerotic (injured) and contralateral (non-injured) arteries withthrombus in rabbits fed with 0.5% cholesterol diet. (A) Representative autoradiogram (HE stain), and immunohistochemistry formuscle actin, rabbit macrophage, GPIIb/IIIa, and fibrin. Immunoreaction is visualized by 3, 3’-diaminobenzidine tetrahydrochloride(brown). Uptake of 18F-FDG is low in the contralateral artery (Upper) comprising smooth muscle cells (SMCs) and some macrophages and a thin layer of mural thrombus comprising platelets and fibrin (arrowheads) covers the luminal surface. Uptake of
18F-FDG is high in atherosclerotic artery (lower rows) that is rich in macrophages. A thicker layer of mural thrombus comprisingplatelets and fibrin (arrows) covers the luminal surface. Immunohistochemical images of GPIIb/IIIa and fibrin correspond to square
frames in the HE images. Bars=500 μm (ARG, HE, SMC, Macrophage) or 100 μm (GPIIb/IIIa and fibrin). (B) Uptake of 18F-FDG and
immunopositive areas for muscle actin, macrophages, GPIIb/IIIa, fibrin, and GPIIb/IIIa and fibrin in arterial sections (n=25 each).
18F-FDG, [18F]-fluorodeoxyglucose.

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YAMASHITA A et al.

Figure 4.   Expressions of nuclear factor κ B (NF-κB) and tissue factor (TF) in atherosclerotic (injured) and contralateral (non-injured)arteries in rabbits fed with 0.5% cholesterol diet. (A) Representative immunohistochemical images of NF-κB and TF show increased
numbers of NF-κB-immunopositive nuclei and TF-immunopositive areas in the atherosclerotic compared with non-atheroscleroticartery. Immunoreaction is visualized by 3, 3’-diaminobenzidine tetrahydrochloride (brown). Bars=100 μm. (B) Representative
double immunofluorescent images for NF-κB and macrophages or smooth muscle cells (SMC) in atherosclerotic arteries. Images
stained with CF488A-labeled anti-NF-κB antibody (green), CF495-labeled antimacrophage antibody (red) or anti-SMC antibody(red), and merged immunofluorescent images. (C) Numbers of NF-κB-immunopositive nuclei and TF immunopositive area (n=25
each), and correlation between NF-κB and TF expression.

11-7055, an inhibitor of inhibitor of κB kinase, significantly
reduced both the number of NF-κB-immunopositive nuclei(Figure 6A) and TF protein production (Figure 6B). Staining
for NADH did not significantly differ among the groups (before culture, 40.4±8.7%; 6 h after culture with DMSO andwith Bay 11-7055, 43.2±10.6% and 46.8±5.0%, respectively;
n=5 each).

Discussion
The main findings of the present study are presented. Significantly more 18F-FDG was taken up and more thrombus formedin the injured than in the non-injured arteries, and 18F-FDG
uptake significantly correlated with thrombus size, NF-κB
immunopositive nuclei and TF expression in the arteries. The
inhibition of NF-κB significantly reduced TF expression inAdvance Publication by-J-STAGE

Arterial 18F-FDG Uptake and Thrombogenicity

Figure 5.  Correlations between 18F-FDG uptake and vascular/thrombus components. Correlation with vascular (A) and thrombus (B)components in arterial sections. Correlation
between 18F-FDG uptake in PET image andthrombus components in corresponding arterial sections (C). 18F-FDG, [18F]-fluorodeoxyglucose; ARG, autoradiography; NF-κB, nuclear
factor κ B; PET, positron emission tomography;
SUV, standardized uptake value.

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YAMASHITA A et al.

Figure 6.   Numbers of nuclear factor κ B (NF-κB)-immunopositive nuclei and tissue factor (TF) protein levels in cultured atherosclerotic plaque. Atherosclerotic lesions (2-mm long) were cultured in conditioning medium with 10% fetal bovine serum. Numbersof NF-κB-immunopositive nuclei (A) and TF protein level (B) in plaques were measured before and 6 h after plaque culture withand without Bay 11–7055, an inhibitor of κB kinase (n=5 each).

cultured plaque tissue.
Arterial 18F-FDG uptake significantly correlated with thesize of thrombus induced by balloon injury of the rabbit iliacofemoral artery (Figure 5B). A clinical study of patients withatherosclerosis has demonstrated higher 18F-FDG uptake inculprit lesions associated with acute coronary thrombosis thanin lesions stented for stable angina.20 Azis et al found significantly more 18F-FDG uptake in rabbit atherosclerotic aortic
segments with than without thrombus formation triggered byRussell’s viper venom and histamine.14 Patel et al reported thatlowering cholesterol absorption reduces plaque burden, 18FFDG uptake and thrombus formation in rabbit atherosclerotic
aortas, whereas 18F-FDG uptake and macrophage content in
the aortas do not correlate.21 Those results imply an association between 18F-FDG uptake and thrombogenic factors in thearteries, but the macrophage content does not necessarily correspond to thrombus formation. The present study identified apositive correlation between 18F-FDG uptake and thrombus
size, as well as TF expression. In addition, not only the areasof fibrin but also those with platelets correlated with 18F-FDG
uptake (Figures 5A,B). Because thrombin generated via the
TF pathway accelerates platelet activation, as well as fibrin
formation,22 evidence of a positive correlation between 18FFDG uptake and TF expression would support previous findings indicating that 18F-FDG uptake is associated with plaquethrombogenicity.
Although radioactivity in thrombus might affect the correlation between thrombus size and 18F-FDG uptake measured
autoradiographically, the amount of radioactivity in the bloodand non-injured artery was equally low. Moreover, thrombus
size also correlated with 18F-FDG uptake measured by PET
imaging (Figure 5C). These results suggest that FDG uptake
reflects the thrombogenic potential of the arterial wall following balloon injury.
Arterial 18F-FDG uptake significantly correlated with macrophage content (Figure 5A). The finding that 18F-FDG uptake
positively correlated with this, but negatively with that of SMC,
is compatible with those of previous studies.8–10,23 Macrophages
and granulocytes express glucose transporter 1 and hexokinase
II in lung inflammatory lesions. 18F-FDG uptake correlates withthe degree of inflammation24 and lipopolysaccharide enhances

18F-FDG uptake by isolated human monocyte-macrophages.25

Therefore, it is considered that 18F-FDG uptake by macrophages is a hallmark of active inflammation in atherosclerotic
plaques. However, clinical and experimental studies have failed
to show a correlation between 18F-FDG uptake and macrophage
content in atherosclerotic lesions.21,26 The evidence suggeststhat the presence of macrophages does not always indicate active inflammation. Recent studies have revealed macrophageheterogeneity in atherosclerotic lesions,27 and approximately
25% of macrophages in coronary atherosclerotic plaques havean antiinflammatory phenotype.28 Moreover, classical but not
innate activation significantly increases 2-deoxy-D-glucose uptake, accompanied by glycolytic pathway activation.29 In thepresent study, CD163-positive cells accounted for only 1% ofmacrophages in rabbit atherosclerotic artery. The positive relation between 18F-FDG uptake and macrophage content in thismodel may be related to a paucity of macrophages with an antiinflammatory phenotype (ie, CD163-positive cells) in the rabbit atherosclerotic artery.
The nuclear transcription factor, NF-κB, affects the inflammatory response, cell proliferation and survival, and angiogenesis in atherosclerotic lesions.30 Activated NF-κB has been
identified in atherosclerotic plaques from humans and in animal models.31–33 The expression of NF-κB in coronary atherosclerotic plaque is more enhanced in patients with unstable
than with stable angina pectoris.34 In the present study, immunoreactivity for NF-κB was distributed predominantly inmacrophages and less so in SMCs in atherosclerotic lesions(Figure 4B), and there was significant correlation among 18FFDG uptake, macrophage content, NF-κB, and TF. These results are compatible with those from a previous report showing colocalization of deoxyglucose and annexin A5, a markerof thrombogenic cell surface, in macrophage-rich atherosclerotic lesions in mice.8 These results also suggest that metabolically active cells in the arterial wall have a high-thrombogenic potential in lesions.
TF is upregulated in both human and rabbit atherosclerotic
lesions4,35 and its expression has been extensively investigatedin cultured monocytic cells, SMCs, endothelial cells and fibroblasts.36 Basal and inducible TF expression is regulated byseveral transcriptional factors, such as Sp1, NF-κB, EGR-1, andAdvance Publication by-J-STAGE

Arterial 18F-FDG Uptake and Thrombogenicity

AP-1. However, its regulatory mechanisms in atherosclerotic
plaques are not fully understood. The present study found that
TF and NF-κB-immunopositive areas were similarly distributed, and a tissue culture study of the plaques revealed that inhibiting NF-κB significantly reduced TF expression (Figures 4,6).
These results indicated that NF-κB mediates TF expression inatherosclerotic plaques. The finding of a positive relationshipbetween 18F-FDG uptake and arterial thrombus size might be
partly explained by TF expression mediated by NF-κB.
Although a prospective clinical study has not yet examined
the predictive value of 18F-FDG-PET in cardiovascular events,
circumstantial evidence has been obtained from a retrospective
series of patients with cancer who underwent PET scanning.
Paulmier et al reported that extensive arterial 18F-FDG uptake
on PET images might be an indicator of an evolving atherosclerotic process in stable cancer patients.37 Rominger et alalso found that increased 18F-FDG uptake in major arteries
emerged as a powerful predictor of subsequent vascular eventsin a large cohort study of patients with cancer.12 In addition, acase–control pilot study suggested that arterial 18F-FDG fociin the aortic arch and carotid bifurcation are associated with arisk of subsequent ischemic stroke.13 Those clinical findings
suggest that 18F-FDG-PET can identify patients at risk for future cardiovascular events and the present results support thisnotion.
The present study did not show balloon catheter-induced
thrombus formation in the normal artery of rabbits fed withconventional diet, because we have previously examined balloon catheter-induced thrombus formation in rabbit femoral
arteries. The thrombus size in the normal femoral artery wassignificantly smaller than that in the injured (atherosclerotic)
artery in rabbits fed with 0.5% cholesterol diets, and did not
differ from that in the femoral artery of rabbits fed with 0.5%cholesterol diet.15
Study Limitations
The thrombus mechanically induced by a balloon catheter does
not replicate spontaneous plaque rupture in human atherothrombosis. Although a recent in vitro study demonstrated that hypoxia augments 18F-FDG uptake in cultured macrophages,38 we
did not assess the contribution of hypoxia to 18F-FDG uptake
in our model. Because thrombus size was evaluated 15 minafter its induction, it was not possible to show thrombus persistence and growth rates, both of which are important factors inhuman atherothrombosis.

Conclusions
Arterial 18F-FDG uptake is associated with thrombogenic fac-

tors and thrombus formation in rabbit atherosclerotic lesions,and arterial 18F-FDG uptake appears to reflect thrombotic riskof atherosclerotic plaques following balloon injury.
Acknowledgments
The work was supported in part by Grants-in-Aid for Scientific Researchin Japan (Nos.23790410 A.Y., 23659573 Y.K., 23390084 Y.A.), Mitsubishi
Pharma Research Foundation (A.Y.), and Integrated Research Project for
Human and Veterinary Medicine, University of Miyazaki (A.Y., Y.A.),
and Project for Developing Innovation Systems from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government(N.T.).

Disclosures
Conflict of Interest: We have no conflicts of interest to declare.

References
1. Sato Y, Hatakeyama K, Marutsuka K, Asada Y. Incidence of asymptomatic coronary thrombosis and plaque disruption: Comparison of
non-cardiac and cardiac deaths among autopsy cases. Thromb Res
2009; 124: 19 – 23.
2. Ueda Y, Ogasawara N, Matsuo K, Hirotani S, Kashiwase K, Hirata
A, et al. Acute coronary syndrome: Insight from angioscopy. Circ J
2010; 74: 411 – 417.
3. Lee KW, Lip GY. Acute coronary syndromes: Virchow’s triad revisited. Blood Coagul Fibrinol 2003; 14: 605 – 625.
4. Drake TA, Morrissey JH, Edgington TS. Selective cellular expression of tissue factor in human tissues: Implications for disorders ofhemostasis and thrombosis. Am J Pathol 1989; 134: 1087 – 1097.
5. Hatakeyama K, Asada Y, Marutsuka K, Sato Y, Kamikubo Y,
Sumiyoshi A. Localization and activity of tissue factor in human aortic
atherosclerotic lesions. Atherosclerosis 1997; 133: 213 – 219.
6. Nicholls SJ, Andrews J, Puri R, Uno K, Kataoka Y. Imaging progression of coronary atherosclerosis. Circ J 2012; 77: 3 – 10.
7. Zagorchev L, Mulligan-Kehoe MJ. Advances in imaging angiogenesis and inflammation in atherosclerosis. Thromb Haemost 2011; 105:
820 – 827.
8. Zhao Y, Zhao S, Kuge Y, Strauss WH, Blankenberg FG, Tamaki N.
Localization of deoxyglucose and annexin A5 in experimental atheroma correlates with macrophage infiltration but not lipid depositionin the lesion. Mol Imaging Biol 2011; 13: 712 – 720.
9. Ogawa M, Ishino S, Mukai T, Asano D, Teramoto N, Watabe H, et al.
18F-FDG accumulation in atherosclerotic plaques: Immunohistochemical and PET imaging study. J Nucl Med 2004; 45: 1245 – 1250.
10. Tawakol A, Migrino RQ, Hoffmann U, Abbara S, Houser S, Gewirtz
H, et al. Noninvasive in vivo measurement of vascular inflammation
with F-18 fluorodeoxyglucose positron emission tomography. J Nucl
Cardiol 2005; 12: 294 – 301.
11. Tahara N, Kai H, Yamagishi S, Mizoguchi M, Nakaura H, Ishibashi
M, et al. Vascular inflammation evaluated by [18F]-fluorodeoxyglucose positron emission tomography is associated with the metabolic
syndrome. J Am Coll Cardiol 2007; 49: 1533 – 1539.
12. Rominger A, Saam T, Wolpers S, Cyran CC, Schmidt M, Foerster
S, et al. 18F-FDG PET/CT identifies patients at risk for future vascular events in an otherwise asymptomatic cohort with neoplastic disease. J Nucl Med 2009; 50: 1611 – 1620.
13. Grandpierre S, Desandes E, Meneroux B, Djaballah W, Mandry D,
Netter F, et al. Arterial foci of F-18 fluorodeoxyglucose are associatedwith an enhanced risk of subsequent ischemic stroke in cancer patients: A case-control pilot study. Clin Nucl Med 2011; 36: 85 – 90.
14. Aziz K, Berger K, Claycombe K, Huang R, Patel R, Abela GS. Noninvasive detection and localization of vulnerable plaque and arterial
thrombosis with computed tomography angiography/positron emission tomography. Circulation 2008; 117: 2061 – 2070.
15. Yamashita A, Matsuda S, Matsumoto T, Moriguchi-Goto S, Takahashi
M, Sugita C, et al. Thrombin generation by intimal tissue factor contributes to thrombus formation on macrophage-rich neointima but notnormal intima of hyperlipidemic rabbits. Atherosclerosis 2009; 206:
418 – 426.
16. Zhao S, Kuge Y, Yi M, Zhao Y, Hatano T, Magota K, et al. Dynamic
11C-methionine PET analysis has an additional value for differentiating malignant tumors from granulomas: An experimental study using
small animal PET. Eur J Nucl Med Mol Imaging 2011; 38: 1876 – 1886.
17. Aide N, Louis MH, Dutoit S, Labiche A, Lemoisson E, Briand M, etal. Improvement of semi-quantitative small-animal PET data withrecovery coefficients: A phantom and rat study. Nucl Med Commun
2007; 28: 813 – 822.
18. Kaim AH, Weber B, Kurrer MO, Gottschalk J, Von Schulthess GK,
Buck A. Autoradiographic quantification of 18F-FDG uptake in experimental soft-tissue abscesses in rats. Radiology 2002; 223: 446 –451.
19. Neumann RA, Knobler RM, Pieczkowski F, Gebhart W. Enzyme
histochemical analysis of cell viability after argon laser-induced coagulation necrosis of the skin. J Am Acad Dermatol 1991; 25: 991 –998.
20. Rogers IS, Nasir K, Figueroa AL, Cury RC, Hoffmann U, Vermylen
DA, et al. Feasibility of FDG imaging of the coronary arteries: Comparison between acute coronary syndrome and stable angina. JACC
Cardiovasc Imaging 2010; 3: 388 – 397.
21. Patel R, Janoudi A, Vedre A, Aziz K, Tamhane U, Rubinstein J, et al.
Plaque rupture and thrombosis are reduced by lowering cholesterollevels and crystallization with ezetimibe and are correlated with fluorodeoxyglucose positron emission tomography. Arterioscler Thromb
Vasc Biol 2011; 31: 2007 – 2014.
22. Crawley JT, Zanardelli S, Chion CK, Lane DA. The central role ofAdvance Publication by-J-STAGE

23.

24.

25.
26.
27.
28.
29.

30.

thrombin in hemostasis. J Thromb Haemost 2007; 5(Suppl 1): 95 –101.
Davies JR, Izquierdo-Garcia D, Rudd JH, Figg N, Richards HK, Bird
JL, et al. FDG-PET can distinguish inflamed from non-inflamed plaquein an animal model of atherosclerosis. Int J Cardiovasc Imaging 2010;
26: 41 – 48.
Mamede M, Higashi T, Kitaichi M, Ishizu K, Ishimori T, Nakamoto
Y, et al. [18F]FDG uptake and PCNA, Glut-1, and Hexokinase-II expressions in cancers and inflammatory lesions of the lung. Neoplasia
2005; 7: 369 – 379.
Deichen JT, Prante O, Gack M, Schmiedehausen K, Kuwert T. Uptake of [18F]fluorodeoxyglucose in human monocyte-macrophagesin vitro. Eur J Nucl Med Mol Imaging 2003; 30: 267 – 273.
Myers KS, Rudd JH, Hailman EP, Bolognese JA, Burke J, Pinto CA,
et al. Correlation between arterial FDG uptake and biomarkers inperipheral artery disease. JACC Cardiovasc Imaging 2012; 5: 38 – 45.
Waldo SW, Li Y, Buono C, Zhao B, Billings EM, Chang J, et al.
Heterogeneity of human macrophages in culture and in atherosclerotic plaques. Am J Pathol 2008; 172: 1112 – 1126.
Boyle JJ, Harrington HA, Piper E, Elderfield K, Stark J, Landis RC,
et al. Coronary intraplaque hemorrhage evokes a novel atheroprotective macrophage phenotype. Am J Pathol 2009; 174: 1097 – 1108.
Tawakol A, Rudd JH, Fayad ZA, Boscá L. Classical but not innate
activation augments 2-deoxy-D-glucose uptake in macrophages: Implications for atherosclerosis imaging. J Nucl Med 2012; 53(Suppl
1): 141.
Monaco C, Paleolog E. Nuclear factor kappaB: A potential therapeutic target in atherosclerosis and thrombosis. Cardiovasc Res 2004; 61:
671 – 682.

YAMASHITA A et al.

31. Brand K, Page S, Rogler G, Bartsch A, Brandl R, Knuechel R, et al.
Activated transcription factor nuclear factor-kappa B is present in theatherosclerotic lesion. J Clin Invest 1996; 97: 1715 – 1722.
32. Hajra L, Evans AI, Chen M, Hyduk SJ, Collins T, Cybulsky MI. The
NF-kappa B signal transduction pathway in aortic endothelial cells is
primed for activation in regions predisposed to atherosclerotic lesion
formation. Proc Natl Acad Sci USA 2000; 97: 9052 – 9057.
33. Chen H, Yang J, Zhang Q, Chen LH, Wang Q. Corosolic acid ameliorates atherosclerosis in apolipoprotein E-deficient mice by regulating the nuclear factor-κB signaling pathway and inhibiting monocyte
chemoattractant protein-1 expression. Circ J 2012; 76: 995 – 1003.
34. Wilson SH, Best PJ, Edwards WD, Holmes DR Jr, Carlson PJ,
Celermajer DS, et al. Nuclear factor-kappaB immunoreactivity is present in human coronary plaque and enhanced in patients with unstableangina pectoris. Atherosclerosis 2002; 160: 147 – 153.
35. Hatakeyama K, Asada Y, Marutsuka K, Kataoka H, Sato Y, Sumiyoshi
A. Expression of tissue factor in the rabbit aorta after balloon injury.
Atherosclerosis 1998; 139: 265 – 271.
36. Mackman N. Regulation of the tissue factor gene. Thromb Haemost
1997; 78: 747 – 754.
37. Paulmier B, Duet M, Khayat R, Pierquet-Ghazzar N, Laissy JP,
Maunoury C, et al. Arterial wall uptake of fluorodeoxyglucose on PET
imaging in stable cancer disease patients indicates higher risk for
cardiovascular events. J Nucl Cardiol 2008; 15: 209 – 217.
38. Folco EJ, Sheikine Y, Rocha VZ, Christen T, Shvartz E, Sukhova
GK, et al. Hypoxia but not inflammation augments glucose uptake inhuman macrophages: Implications for imaging atherosclerosis with
18fluorine-labeled 2-deoxy-D-glucose positron emission tomography. J Am Coll Cardiol 2011; 58: 603 – 614.